Dormancy is a phenomena which is found in representatives of the plant kingdom as well as the animal kingdom.
The germination of various grains and seeds comprising the necessary propagation organs is delayed under certain circumstances and yet the grains or seeds are capable of germinating after various periods of time. The period of time in which germination of such seeds may be delayed varies and depends both on intrinsic properties of the seed as well as on the nature and extremity of the environmental conditions. Seeds have been shown to be in dormancy for a few days, a year, several years and even for more than several centuries (as was discovered lately in the case of some nympheaceae and seeds of trees of the Leguminosae family (Shen-Miller, J., et al., American Journal of Botany, 82:1367-1380, 1995)).
In some cases, the capability to develop dormancy lies in the embryo envelopes. In such a case, the separation of the envelopes from the embryo, result in its immediate termination.
In other cases, chemical growth inhibitors capable of preventing termination are present in the embryo itself and thus even a bare embryo may remain dormant (such as in the case of Rosaceae plants such as Kerria, Peach, etc.).
In plants, a state of dormancy may be found in the whole plant or in one or more of its parts. Dormant plants are plants which have two main metabolic states in their growth cycle. In their dormant state, the plants' metabolism is extremely low, and the plant growth process is significantly inhibited although differentiation of certain cells may occur. In their active state, the plants' metabolism rate is higher, the cells divide and differentiate and there is significant growth of various parts of the plant. In some cases, the whole plant enters the dormant state. Such is the case in Narcissus plants in which during the dormant state the only remaining viable part is the bulb which is in its dormant state. In other cases, some parts of the plants may be active while other parts may be in dormancy such as, for example, is the case of apple trees.
Substances capable of inhibiting germination have also been shown to be present in the juice of fleshy fruits or in other plant organs which produce juice. Examples are tomatoes, grapes, kiwi, watermelon and grapefruit wherein pips present in the fruit do not germinate although their surroundings are suitable for germination due to the water within the fruit.
Several plant-derived substances having an effect on cell proliferation have been reported. For example, European Patent Application No. 0381514 describes compositions comprising both naturally derived as well as synthetically prepared sphingolipids which have growth inhibitory activity on various kinds of cells. Another well known plant-derived substance having an anti-mitotic effect on various kids of human cells is the substance colchicine (Samson, F. E., A. Rev. Pharmac. Toxic 16:143 (1976)). The Narcissus alkaloid pretazettine, was shown to have a cytotoxic effect on Rausher virus-carrier cells as well as anti-leukemic activity in leukemic mice although the predominant activity of the substance was shown to be an antiviral activity (Furusawav. E. et al., Chemotherapy, 26:36-45, (1980) and Furusawa, E. et al., Proc. Soc. Exp. Biol. Med. 152:186-191, (1976)). Ulex europaeus seed extracts were shown to comprise a non glycoprotein lectin capable of reversibly inhibiting growth of certain lymphocytes as well as to inhibit the growth of various reticulo endothelial tumor cell lines (Pirofsky, B., et al., Vox-Sang, 42:295-303, (1982) and Pirofsky, B. et al., J. Biol. Response Mod, 2:175-185, (1983)). Root extract of Panex ginseng was shown to decrease DNA synthesis measured by [H3]-thymidine incorporation of V 79 Chinese hampster lung cells. Another substance, Narciclasine obtained from bulbs of various Narcissus varieties was shown, amongst other of its activities, to inhibit growth of wheat kernal radicals (Ceriotti, G., et al., Tumors 53:359-371 (1967)). Bulbs of Pancratium littoral collected in Hawaii were found to contain a product designated pancratistatin capable of inhibiting growth of various neoplastic cell lines in vitro (Pettit. G. R., et al., J. Nat. Prod, 49:995-1002 (1986)).
Against this, many plant extracts having an opposite effect on cells, i.e., capable of augmenting their proliferation were also described such as, for example, the methanolic extract from the root of Scutellaria baicalensis georgi were shown to significantly augment the cellular activity of fibroblasts (Chung, C. P., et al., Planta-Med, 61:150-153, (1995)). Gibberellin-like growth substances were found in six different plant species having bulbs (Staby, G. L., Hort. Science, 399-400 (1970)). Several cytokinins which were found in roots that developed from Narcissus bulbs had an effect on bulb growth of the plants in which they were detected (Vanstaden, J. V., Pflanzenphysiol., 86:323-30 (1978)).
The phenomena of dormancy may also be found in the animal kingdom, for example, in the small crustacean Artemia salina (Finamore and Clegg In: The Cell Cycle, Academic Press Ed., 249-278, 1969). The natural environment of this marine crustacea is usually briny ponds. After fertilization, the early stages of development of artemia involve the formation of a blastula which then becomes a gastrula. Under severe environmental conditions such as dehydration (drought), the gastrula is capable of forming a cyst wherein the whole organism enters a dormancy phase. The dormant artemia gastrula (commonly miscalled “artemia eggs”) are capable of remaining in their dormant state for many years. When the encysted gastrula are rehydrated, the various metabolic activities of the artemia are resumed and protein synthesis can be seen after about 10 minutes. However, DNA synthesis and cell division are absent until about after 60 hours (Le Gal, Y, In: Biochimie Marine, (Ed. Masson) p. 176, 1988).
Various plant derived compositions (such as retinoic acid (U.S. Pat. No. 5,438,073) and α-hydroxy acids (Ditre, C. M., et al., J. Am. Acad. Dermatol., 34:187-195, 1996)) as well as animal derived extracts have been proposed for use in the cosmetic field for stimulating the proliferation and renewal of epidermal cells. Such compositions were considered to be useful in the cosmetic field where it is accepted that the natural renewal process of epidermis is slowed down with aging. It is believed that removal of the outer surface with simultaneous stimulation of growth of new cells in the inner layers of the epidermis to divide and migrate to the outer surface, will result in skin renewal and in a younger skin appearance. However, it is also known and has been recently shown that the increase in cell division is a crucial factor in converting normal cells into premalignant or malignant cells (Ames, B. N. et al., Environ. Health Perspect 101:35-44 (1993)).
It is also believed today that normal human and animal cells have a finite capacity to replicate. It has been shown that the number of mitotic events that cultured normal animal cells can undergo appear to be inversely related to the age of the donor from which they were obtained (Hayflick, L., Clin. Geriatr. Med., 1:15-27, (1985)). It has also been shown that cell cultures obtained from patients with accelerated aging syndromes undergo less replications than cell cultures obtained from age matched control individuals.
Glossary
The following is the meaning of some terms of which will be used in the text below:
Dormancy—a state in which there is a marked decrease in the metabolic rate of cells or tissues resulting in the inhibition of growth and proliferation of the cells or tissue.
Dormans—substances naturally found in cells or tissue and which are capable of inducing cells or tissue to enter a state of dormancy or of maintaining the dormant state in cells or tissues that have already entered that state. The dormans may be obtained from a variety of plant parts which are capable of entering into a state of dormancy; from juice of various fruits which contain dormans capable of inhibiting germination of seeds within the fruit; from animals which are capable of entering a phase of dormancy in their life cycle, e.g. gastrula of certain crustaceans such as artemia or dafnia; etc. In some cases: the dormans are found within a dormant tissue, e.g. in a dormant seed or in the gastrula of artemia or dafnia; in other cases the dormans are found in a tissue surrounding the dorman tissue or organ, e.g. in a fruit juice surrounding a dormant seed.
Extract—At least one substance obtained by any of a variety of extraction methods known in the art. For example, the extract may be an aqueous extract, a glycolic extract an alcoholic extract, an oily extract, etc. The extract in accordance with the invention is obtained from cells or tissues from a part of a plant or animal capable of entering a state of dormancy. The cells or tissue may be obtained directly from the plant or animal and the extract may then be prepared therefrom. Alternatively, cell cultures may first be prepared from the plant or animal cells or tissues and then the cell cultures may be grown for various periods of time. In order to prepare an extract, the cells are then harvested from the cell cultures, the cells and their growth medium are separated and an extract may be prepared either from the cells themselves or from the growth medium (which will be referred to as “supernatant”) which contains substances secreted by the cells into their growth medium. Thus, the “extract” may be obtained directly from plant or animal tissue or from an animal or plant cell or tissue culture.
Dorman extract—an extract obtained from a plant cell or tissue, from fruit or from an animal cell or tissue which comprises dormans.
Enriched dorman preparation (EDP)—a preparation derived from a natural source which comprises dormans in a concentration larger than that which is found in a natural unprocessed extract. The EDP may be obtained by purification of a natural extract to obtain fractions which contained dormans in the larger concentration, e.g. by various chromatographic techniques, by filtration, etc., as well as by biological means including growing cells or tissue which are capable of producing dormans under conditions in which they produce dormans in relatively large quantities and collecting their secretion products. In order to determine whether a preparation is an enriched dorman preparation, the preparation may be assayed for a specific biological activity associated with dormans, as described below. EDP contains a substantially higher concentration of dormans as compared to the natural preparation, e.g. at least 1.5 folds, preferably 2 folds and typically at least 2.5 folds to the concentration of the dormans in the natural preparation.
Producer cells or producer tissue—cells or tissue which are capable of producing dormans which may thus be extracted therefrom.
Target cells or target tissue—cells or tissue which are contacted with dormans, in accordance with the in invention and which thereby enter a state of inhibition of their growth or proliferation or maintain such a state as the result of contact with dormans.
Dorman analog—a substance, typically synthetic, which has a dorman-like activity in that it is capable of inducing dormancy in the same cells or tissue induced to dormancy by the dorman and which in accordance with the invention, is also capable of inhibiting growth and proliferation of target cells or tissue.
Dorman composition (DC)—a composition comprising, as active ingredient, an amount of dorman (e.g. as a dorman extract) or dorman analog effective in inhibiting growth and proliferation of target cells or tissue (“effective amount”). A dorman composition may comprise a naturally derived EDP, a composition comprising synthetic dormans as well as dorman analogs.
Active Extract (AE)—extracts obtained from cells or tissues from a part of a plant or animal capable of entering a state of dormancy during the non dormant state.